The goal is to identify a protein species synthesized by human chondrosarcomas, and released into plasma; to develop methods for determining the serum concentration of the protein, which can be used preoperatively in the initial diagnosis of chondrosarcoma and postoperatively to detect local recurrence or metastasis. Protein species extracted from human chondrosarcomas have been separated from proteoglycans by density gradient centrifugation and identified in terms of their molecular weights SDS-PAGE. A 15,000 MW protein is present in relatively high concentration in matrix protein fractions from all human chondrosarcomas and from some benign cartilage neoplasms. The 15K protein was purified by chromatography on DEAE cellulose in 6 M urea and on Sephacryl S200 in 4 M GdmC1, and an antiserum to the protein was prepared. In parallel, a 35,000 MW protein was isolated from bovine fetal epiphyseal cartilage and characterized and an antiserum was prepared to the 35K protein. The immunofluorescent localization of the 35K protein was then demonstrated in cartilage growth plate. The 35K protein has a high affinity for hydroxyapatite, and is selectively concentrated in exactly the same region where mineralization occurs, suggesting that the 35K protein is involved in the mineralization which occurs during endochondral ossification. Using the antiserum to the 15K protein, the immunological relation between the two proteins was examined. The antiserum to the 15K protein did not react with the native undenatured 35K protein. However, when the 35K protein was denatured, subjected to SDS-PAGE and examined by Western blotting, the antiserum to the 15K protein reacted strongly with the 35K protein. These observations suggest that the 15K protein is immunologically related to, and probably represents a fragment of the 35K protein present in developing normal epiphyseal cartilage and in the cartilage growth plate.